Fresh and cultured buccal cells as a source of mRNA and protein for molecular analysis

Abstract

We developed a method for obtaining viable buccal cells from mouthwash samples for use as a source of mRNA and protein. Immunofluorescent analysis showed that most cells were derived from nonkeratinized parabasal epithelia, with a minor proportion of proliferative cells. Gene expression was detected in buccal cells using reverse transcription PCR, Western blot analysis, and immunofluorescence. Using a keratinocyte-specific medium, buccal cells could be cultured on Matrigel-coated permeable filters for up to 2 weeks while maintaining the expression of some epithelial-specific markers, including cytokeratin 13, cytokeratin 10, transferrin receptor, and beta-integrin. The basal marker cytokeratin 14 and Ki67, an indicator of cellular proliferation, were detected in a few cells. We show that buccal cells can be obtained from a noninvasive procedure for use as a source of material for biochemical analyses. A population of the buccal cells can be maintained in culture for up to 2 weeks using keratinocyte-specific medium in combination with extracellular matrix.

Protein and RNA extraction from mucosal swabs: a minimally invasive source of ecological data for studies of natural populations

“…Specifically, mucosal swabs allow for the quantitation of antibodies (Brandtzaeg ; Weissenbacher ), epithelial and immune cell culture (Michalczyk et al . ), DNA and RNA extraction (Spivack et al . ; van Wieren‐de Wijer et al .…”

“…Transcripts of these genes have been detected in mucosal epithelium in other species (see Michalczyk et al . ; Spivack et al . ; Park ; Dietz et al .…”

“…To explore the potential downstream application of mucosal swabbing, cDNA samples were used to PCR-amplify housekeeping genes hprt, rps5, rps19 and gapdh, and two selected immune genes: interleukin 1a (il1a) and interleukin 8 (cxcl8). Transcripts of these genes have been detected in mucosal epithelium in other species (see Michalczyk et al 2004;Spivack et al 2004;Park 2006;Dietz et al 2011). We used the following primers pair: hprt (5 0 -AGCTTGCTGGTGAAAAGGAC, 5 0 -TTAAGTCAAGGGCATATCC), gapdh (5 0 -TGTCCCCACCCC-CAATGTATC, 5 0 -TCCGATGCCTGCTTCACTACCTT), rps5 (5 0 -TCACTGGTGAGAACCCCCT, 5 0 -CCTGATTCACACGGCG-TAG), rps19 (5 0 -CCTTCCTCAAAAAGTCTGGG, 5 0 -GTTCTCAT CGTAGGGAGCAAG), il1a (5 0 -RAAGAAGAGACGGTTGAGT T, 5 0 -TTGAGGTTGTCATTMAGGAT), cxcl8 (5 0 -CACRCCTTTC-CATCCCAAA, 5 0 -GGCACACCWCATTTCCATT).…”

Coleta, transporte e armazenamento de amostras para diagnóstico molecular

“…Para análise de DNA, amostras coletadas em swabs específicos podem ser secas e transportadas a temperatura ambiente. Amostras de bochecho podem ser transportadas a temperatura ambiente e são estáveis por até uma semana (7,10,22) .…”

Extracting, storing and distributing DNA for a birth cohort study

“…Access to tissues other than blood is more difficult. One possibility is to access epithelial cell types by sampling buccal mucosa 20 . Another would be skin‐punch biopsies to provide fibroblasts, though these may well be deemed to be unacceptable, especially in children.…”